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We developed a new method for studying the process of thrombus formation. This entailed combining a technique for triggering thrombus formation by inducing ROS（reactive oxygen species） production with a laser and our unique live tissue imaging technique. Using this approach to observe various animals carrying genetic modifications causing abnormal platelet function, we clarified the relationship between platelet function in vitro and thrombus formation in vivo. As shown in Figure 2, ROS（reactive oxygen species）-triggered thrombus formation was highly reproducible. This is the first time thrombi have been visualized with sufficient resolution to identify individual platelets.
In our model, thrombi were constructed by platelets that maintained an oval form, and no injury to the endothelium was observed. From the observation of various knockout and chimeric mice deficient in one or more inflammatory cytokines, it is apparent that inflammatory cytokines contribute to the expression of von Willebrand factor (vWF) by endothelial cells under ROS（reactive oxygen species） stimulation. In addition, IL-1 and IL-6 also contribute to thrombus formation. These inflammatory cytokines affect the endothelium and increase thrombus stability in co-operation with integrin signaling. Association between inflammation and thrombus formation has been suggested in many reports. Our analysis showed that inflammatory cytokines signaling in the endothelium contributes to thrombus formation.
In recent years, there have important advances in the research on pluripotent stem (ES, iPS) cells, and clinical application is expected in a wide range in regenerative therapies. To translate the basic research findings to clinical practice, one must know how cells generated in vitro function within a normal in vivo environment and in animal models of disease before attempting to apply them to human subjects. However, no methods existed to characterize iPS-derived cell dynamics in vivo. We visualized iPS-derived platelet dynamics in vivo in collaboration with the team of Prof. Eto at the Center for iPS Cell Research and Application. iPS-derived platelets circulated in the mouse bloodstream and formed thrombi after laser irradiation in cooperation with endogenous mouse platelets. Our findings confirmed that iPS-derived platelets could circulate in the mouse bloodstream and had the ability to form thrombi just as endogenous platelets do. We suggest our imaging technique would to be very useful for testing the safety and usefulness of clinical applications that use iPS-derived cells.
Resent advances in micro-devices have enabled us to further analyze the function of iPS-derived platelets. For example, we coated a flow chamber with vWF or cultured endothelial cells and flushed platelets loaded with a Ca2+ indicator through the chamber. Using this approach, we found that in both normal platelets and iPS-derived platelets, the intracellular Ca2+ concentration increases upon attachment to the micro-chamber. Devices that mimic the reaction in vivo have also been developed, and further development is expected.